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Administration of recombinant interleukin-12 (rIL-12) into CSA1M fibrosarcoma-bearing mice results in complete regression of growing tumors. This tumor regression is associated with massive lymphoid cell infiltration to tumor sites and is completely blocked by injection of anti-interferon-gamma (IFN-gamma) monoclonal antibody (mAb). We investigated whether anti-IFN-gamma mAb exerts its suppressive effect on tumor regression by blocking the IL-12-induced lymphoid cell migration to tumor sites or by inhibiting the secondary effects of IFN-gamma produced by infiltrating cells. Injection of anti-IFN-gamma mAb to CSA1M-bearing mice before IL-12 treatment prevented the induction of tumor regression, whereas this treatment affected only marginally the infiltration of lymphoid cells to tumor masses. In accordance with this, IFN-gamma mRNA was expressed inside tumor masses by infiltrating cells after IL-12 therapy irrespective of whether anti-IFN-gamma mAb was injected. However, anti-IFN-gamma mAb treatment almost completely abrogated the in situ expression of inducible nitric oxide synthase (iNOS) as well as IFN-inducible protein-10 (IP-10) genes as examples of IFN-gamma-inducible genes. Immunohistochemical analyses also revealed that the expression of iNOS protein was completely inhibited by anti-IFN-gamma injection. These results suggest that the implementation of in situ IFN-gamma activity and its secondary induction of anti-tumor pathways such as iNOS and IP-10 expression are important processes in the IL-12-induced tumor regression.

We previously demonstrated that proliferation of terminally differentiated Th1 clones depends primarily on an interleukin-12 (IL-12)-paracrine mechanism mediated by their interactions with antigen-presenting cells (APC) rather than on an IL-2-autocrine mechanism. Such a Th1 clone (4-86, C57BL/6 origin) was cultured with recombinant IL-12 (rIL-12) in the absence of either antigen or APC. Some cells survived for several passages of culture with only rIL-12, and by limiting dilution, several clones highly reactive to rIL-12 alone were obtained. One of these clones, designated 2D6, was found to proliferate strongly in response to less than 1 pg/mL of rIL-12. This clone exhibited the following surface phenotypes: CD3+, T cell receptor (TCR) alpha beta+, Vbeta11+, NK-1.1-; CD4-CD8-; LFA-1+, ICAM-1+; and CD28+, CD80+, CD86+, CTLA-4-. In accordance with high responsiveness to IL-12, 2D6 cells were also found to express IL-12 receptor (IL-12R) as detected by incubation with rIL-12 and then staining with anti-IL-12 monoclonal antibody (mAb). Stimulation of 2D6 with rIL-12 resulted in the expression of interferon-gamma (IFN-gamma) and IL-10 mRNAs and production of these cytokines. The 2D6 clone responded to IL-2 (vigorously), IL-7 (moderately), and IL-4 (mildly) in addition to IL-12. However, the Ab capture assay using anti-IL-12 mAb enabled us to quantify IL-12-specific activity contained in a given sample. Thus, this study describes the unique features of the IL-12-responsive T cell clone and demonstrates the utilization of this clone in the quantitation of a specific IL-12 activity.

We previously purified macromolecular-translocation inhibitor-III (MTI-III), which inhibits the binding of the activated glucocorticoid-receptor complex (GR) to nuclei, to homogeneity from rat liver, and we found that the purified MTI-III bound to partially purified activated GR under low salt conditions at slightly acidic pH [Liu, G., Okamoto, K., and Isohashi, F. (1993) Eur. J. Biochem. 218, 679-687]. This was the first direct evidence that the inhibitor acts through a direct interaction with the activated GR. In this study, we examined whether the purified MTI-III could interfere with the binding of GR to a DNA fragment containing a specific glucocorticoid-response element (GRE). Under nearly isotonic salt conditions at neutral pH, the activated GR bound to the GRE but not to nonspecific DNA. Under similar conditions, the activated GR also bound to the purified MTI-III. The resulting GR/MTI-III complex did not bind to the GRE. We also found that addition of MTI-III to the GR/GRE complex resulted in time-dependent disruption of the GR/GRE complex and formation of the GR/MTI-III complex. The half-life of the GR/GRE complex in the presence of MTI-III was about 13 min. These results suggest that MTI-III enhances the release of GR from the GR/GRE complex and immediately forms a stable GR/MTI-III complex.

We previously found a novel endogenous factor in rat liver cytosol, named ATP-stimulated glucocorticoid-receptor-translocation promoter (ASTP), that increased the binding of activated glucocorticoid-receptor to nuclei in the presence of ATP. In this work, we immunized rabbits with the purified ASTP protein and characterized the antibodies with regard to titer, cross-reactivity and specificity. An IgG fraction from sera of the immunized rabbits contained specific antibodies to ASTP. The anti-ASTP IgG could precipitate the ASTP protein without the activity. Immunoblot analysis revealed a major band of 48 kDa in rat liver cytosol that migrated to the same position as the purified ASTP protein by SDS-PAGE, and an additional minor band of about 50 kDa. Monospecific antibodies purified from the IgG fraction using the antigen (the purified 48-kDa ASTP protein) immobilized on a polyvinylidene difluoride membrane also reacted with both the 48-kDa ASTP protein and the 50-kDa protein in rat liver cytosol, suggesting that this 50-kDa protein is immunologically related to the 48-kDa ASTP protein. Densitometric quantification of immunoblots demonstrated that the rat kidney cytosol contained ASTP protein at a concentration of about 20% of that of liver cytosol. Other tissues such as brain, skeletal muscle, heart, and lung, contained neither the ASTP protein nor the activity.